1. Field of the Invention
The invention of this application relates to an effective and highly efficient method for introducing a random mutation in which mutation can be efficiently introduced into a cell or an organism individual and also a risk of extinction of treated cell or individual groups can be reduced, and this invention also relates to mutants and mutated phenotypic gene obtained by the said method.
2. Description of the Related Art
With regard to an art for a genetic modification of cells or organism individuals, a method where mutagen such as ultraviolet ray, radioactive ray or mutagenic substance is applied to cells or organism individuals, a method where exogenous gene is introduced into cells or organism individuals to modify by means of genetic engineering, etc. are available. In the case of induction of mutation in specific gene, there has been known a method where genetic engineering means such as site-specific mutation induction and in vitro mutation induction by accumulation of replication mistake in DNA utilizing a PCR amplifying technique.
Generally, when the site into which gene or mutation to be modified is clear, genetic engineering means may be sometimes effective while, when the knowledge about the phenotype to be modified or gene therefor is insufficient, there is an effective method utilizing a random mutation induction where mutation is randomly introduced into gene and, from the resulting mutant, cells or organism individuals having the desired mutation phenotype is selected. In inducing the random mutation, a method where mutation is induced by irradiation of ultraviolet ray, X-ray or radioactive ray to cells or organism individuals, a method where mutation is induced by treating with a mutagenic substance such as nitrogen mustard or nitrosoguanidine, and the like are available.
In the conventional art for introducing a random mutation, the mutation rate induced by ultraviolet ray or mutagen has an important influence on efficiency and effect of the treatment. Thus, when the induced mutation rate is within an optimum range, mutation in a sufficient amount for DNA is accumulated while, when it is less than the optimum amount, mutation may be sometimes repaired by a repairing mechanism, etc. of DNA whereby mutation cannot be introduced efficiently. Further, when it is more than the optimum amount, the lethal effect to the organism by the introduced mutation becomes strong whereupon, before the desired mutant is obtained, the group into which mutation is introduced and is treated therewith dies out resulting in no production of the desired mutant.
The same thing may be said for the optimum amount not only just for one treatment but also for plural and continued treatments where mutagenetic treatment and mutant selection are carried out one after another in order to obtain more highly useful mutant. Thus, unless the optimum amount is determined carefully, the efficiency is bad or the group subjected to a mutagenesis dies out whereby it is at last impossible to obtain a highly useful mutant. In addition, there may be a case where it is necessary to introduce plural mutations into a gene in which phenotype of cells or organism individuals to be modified by a random introduction of mutation is single or a case where it is necessary to introduce mutation into plural genes and, in such cases, randommutation is to be inserted until preferred mutation is accumulated in such genes. However, accumulation of many mutations results in a high risk where lethal mutation is introduced into the gene which is necessary for living. Thus, the higher the mutation rate, the more the risk of extinction of the treated cells or organism individuals whereby it cannot be expected to obtain a useful mutant in an efficient manner.
In recent years, although the object is not to genetically modify the cells or the organism individuals, a method has been developed for an efficient introduction of a random mutation into gene inserted into a plasmid in the following mutant of Escherichia coli utilizing the fact that the mutation rate of E. coli simultaneously having mutD, muS and mutT (which are mutator genes concerning proofreading mechanism of mispair of DNA base pair, A/T-G/C transversion and mismatch repair of DNA, respectively) is 5,000-fold of a wild type strain (Molecular Biotechnology, 7:189-195, 1997). According to such a method, it is possible to introduce a mutation for each 1,000 base pairs into genes on plasmid by incubation for 24 generations within about one day. However, although such a high mutation rate increases the probability of mutagenesis of gene into which mutation is to be introduced, that also increases a risk of introduction of mutation into other genes such as those necessary for living. Therefore, when the length of the DNA region into which mutation is to be introduced is 100 base pairs or less or when mutation is to be introduced into plural parts, a PCR is recommended because the above method is not practical due to an increase in numbers of growth generation. It has been also pointed out that, since the mutation rate is high, incubation of the strain for a long period causes an affection to the cell per se or genotype thereof whereby carefulness is required. Accordingly, although the said method is suitable for introduction of mutation into gene on plasmid introduced into a host, it is not suitable for a genetic modification of the host itself.
As mentioned above, in the conventional method for the introduction of a random mutation into cells or organism individuals, introduction of many mutations and avoidance of extinction of mutation-introduced group are in a relation of antinomy whereby it is difficult to obtain various and useful mutants in an efficient manner.
The invention of this application has been achieved in view of the above circumstances and its object is to provide a method where a random mutation is introduced into a cell or an organism individual in a high mutation rate and, at the same time, risk of extinction of treated group is reduced and useful and various mutants are efficiently obtained.